KR100835747B1 - Mre11 protein relating to aging and methods of controlling aging by using it - Google Patents

Abstract

The present invention relates to an aging-related Mre11 protein and an aging control method using the same, and relates to an anti-aging method, an anti-aging screening method, and an anti-aging agent using the Mre11 protein which decreases as aging progresses.

The present invention also relates to a method for promoting aging, a method for screening an aging accelerator, and an aging accelerator.

Mre11, aging

Description

Mre11 protein relating to aging and methods of controlling aging by using it}

1 shows protein expression of Ku70 and Mre11 in CD4 + T cells obtained from 48 volunteers from 20 to 80 years of age.

Top panel; The total protein amount of Ku70 (A) or Mre11 (B) isolated from human lymphocytes was measured by Western blot using rabbit monoclonal antibodies against human Ku70 or human Mre11. The 48 samples analyzed showed 5-6 protein expressions at each age group (20-30, 40-50, 60 and above). I.S indicates internal standards obtained from Ku-expressing cell lines. 1, 4.5 and 20 μg of digests obtained from the A549 human lung cancer cell line were loaded on the gel.

Lower panel: Scatter plot of Ku70 (A) or Mre (B) protein levels over age From several different elderly, percent protein of Ku70 or Mre11 was divided by the 20 μg internal standard shown above.

2 shows a correlation analysis of Ku70 and Mre11 expression in people between 20s and 80s. The variance plot of Ku70 and Mre11 expression shows a meaningful primary correlation between Ku70 and Mre11. (Pearson's correlation coefficient, r = 0.5472, p <0.0001)

In human somatic cells, telomeres lose their sequence as they replicate at the ends of the DNA and gradually shorten during cell division. Maintenance and stabilization of telomere DNA is supported by the binding of several DNA-binding proteins, including those involved in double strand break (DSB) repair. Since functional telomeres are essential for continuous cell proliferation and stable inheritance, loss of end capping of chromosomes is important for both aging and cancer (Refs. 19-21) .DSBs lose genetic information if not properly repaired. It can be a disaster part that can be discarded, mutated or killed, In mammals, two large pathways exist to repair DSB: homologous recombination (HR) and non-homologous end- Nonhomologous end-joining (NHEJ) pathway (Ref. 22) Ku70 exists as a heterodimeric complex of Ku70 / ku80 and is a component of DNA-dmlwhs protein kinase (DNA-PK). Together with PKcs (DNA-PK catalytic subunit), Ku70 is involved in DSB repair via NHEJ DNA-PK Mutations in certain components result in end-terminal fusions between chromosomes and harmful terminal rearrangements (Ref. 23) Similarly, telomeres fusion and inactivation have been reported to occur when the Ku70 gene is lost in mice (Refs. 23, 24) Mre11 exists in the form of a complex with Rad50 and Nbs1. The complex affects recombinant DNA repair by promoting recombination between the sister chromosomes, a partial hypomorphic mutation of Mre11 caused human genetic instability disease (Ref. 25).

Aging is the process by which healthy people change to weaker ones, which decrease the performance of most physiological systems and increase their susceptibility to disease and death. Aging and maximum human life are species dependent. This must be at least genetically controlled (Refs. 26, 27). Up to 25% of mutations in human lifespan are genetic (Refs. 28, 29) and the rest is due to environmental exposure, accidents and injuries, and chance. Very long lives up to 90 years of age appear to have an even more genetic basis (Ref. 30) and explain whether people over 100 or near age 100 are clustered into families. On the other hand, progeroid syndrome, which promotes aging and death at a young age, has been found to have a genetic cause (Refs. 31 and 32).

Indeed, several regions of chromosomes 1, 2, 4, 7, 12 and 17 interact with each other and with environmental factors between them, affecting life expectancy.

The inventors have found that the total protein of Mre11 decreases with age in human lymphocytes and completed the present invention.

It is an object of the present invention to provide a method for preventing aging, an antioxidant screening method, and an anti-aging agent.

An object of the present invention is to provide a method for promoting aging, a method for screening an aging accelerator, and an aging accelerator.

For this purpose, the present invention provides an anti-aging method for animals (except humans), characterized by increasing intracellular Mre11 protein.

More specifically, the method of increasing the Mre11 protein is an anti-aging method of an animal characterized in that the delivery of the Mre11 protein into the cell and / or an anti-aging method of the animal, characterized in that to promote the transcription of Mre11 protein in the cell. to provide.

Mre11 protein in the present invention includes functional equivalents and functional derivatives of Mre11 protein. “Functional equivalents” include amino acid sequence variants in which some or all of the naturally occurring protein amino acids are substituted, or some of the amino acids are deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a portion not directly involved in the physiological activity of Mre11.

A "functional derivative" refers to a fragment of the platelet derived growth factor polypeptide, a protein comprising the fragment, or a protein that has been modified to increase or decrease the physicochemical properties of the protein. The term "fragment" refers to an amino acid sequence corresponding to a portion of a protein and having a common origin element, structure and mechanism of action that falls within the scope of the present invention. Modifications to alter the stability, shelf life, solubility, etc. of Mre11, or derivatives that have been modified to alter the relationship with, for example, a substance that interacts with Mre11, are also within the scope of the present invention. Methods of making functional derivatives of such proteins are known.

Another form of the invention provides a pharmaceutical composition with an anti-aging agent, characterized in that it comprises an intracellular Mre11 protein. The pharmaceutical composition may be prepared and administered in various formulations and methods.

For example, oral administration can be mixed with an inert diluent or an edible carrier, sealed in a hard or soft gelatin capsule, pressed into tablets or administered directly in the diet. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

The tablets, troches, pills, capsules and the like may be used in combination with a binder such as gum tragacanth, acacia, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid and the like; Lubricants such as magnesium stearate; And sweetening agents such as sucrose, lactose or saccharin; Or flavoring agents such as peppermint, winter green oil or cherry flavor. If the dosage unit form is a capsule it may contain a liquid carrier in addition to the above substances. Various other materials may be added that change the physical form of the coating or dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. Elixir syrups may contain sucrose as a sweetener, methylparaben and propylparaben as a preservative, dyes and flavoring agents such as cherry or orange flavors with the active compound. All materials used to prepare any dosage unit form must be pharmaceutically pure and substantially nontoxic in the amounts used. In addition, the active compounds may be prepared in sustained release formulations.

For oral administration, the compositions of the present invention can be used in the form of non-ingestive mouthwashes and toothpastes in admixture with excipients. Mouthwashes can be prepared by adding the active ingredient in the required amount to a suitable solvent, such as sodium borate solution. Alternatively, the active ingredient may be added to an antiseptic cleaner containing sodium borate, glycerin and potassium bicarbonate, or dispersed in toothpaste in the form of gels, pastes, powders and slurries, or water, binders, abrasives, flavoring agents, foaming agents and It may be added in a therapeutically effective amount to a paste toothpaste that may include a humectant.

Various formulations, such as for other injectable and parenteral administration, can be prepared according to the techniques described or commonly used in books well known in the art (eg, "Remington's Pharmaceutical Sciences" 15th Edition).

Dosages can vary depending on the condition of the treated patient, and the person in charge can determine the appropriate dose for each patient. In addition, for human administration, the preparations must meet sterile, pyrogenic, general safety and purity criteria in accordance with the Agency.

Another form of the invention comprises the steps of treating any test substance with a test cell; Measuring the content of Mre11 protein in the cells of the test cell; And it provides an antioxidant screening method comprising the step of selecting a substance that increases the content of intracellular Mre11 protein.

Methods of measuring protein content include electrophoresis of proteins, measurement by comparison with standard samples, measurement using specific antibodies, measurement by producing arbitrary reporters and fusion proteins, and the like.

As a method for measuring the transcription amount of Mre11 protein, a recombinant vector having a Mre11 regulatory promoter may be used.

The method of confirming the expression of a promoter can be performed using a reporter gene. Reporter genes are not limited to specific genes. Any gene that expresses a product that can be easily measured can be used. Suitable reporter genes are well known to those skilled in the art. Examples of reporter genes include other enzyme detection systems such as CAT (chloramphenicol acetyl transferase), luciferase, beta-galactosidase; Bacterial luciferase, alkaline phosphatase and green fluorescent material (GFP) are included, but are not limited to these. Detection of reporter genes is well known to those skilled in the art. When a reporter gene is detected by its enzymatic activity, it generally comprises providing the enzyme with its appropriate substrate and detecting the reaction product (eg light generated by luciferase). Detection can include simply detecting the presence or absence of the reporter gene product. Another method may include quantification of the expression level of the reporter gene product. The method of quantification can be absolute quantification or can be quantified compared to the expression level of the housekeeping gene. Such detection may be performed manually or may be performed automatically as in a high-throughput screening system.

High throughput test methods for the presence, absence or quantification of gene expression are well known to those skilled in the art. For example, US Pat. No. 5,559,410 describes a high throughput screening method for proteins, and US Pat. No. 5,585,635 describes a high throughput screening method for nucleic acid binding. U.S. Patents 5,576,220 and 5,541,061 also describe high throughput screening methods for ligand / antibody binding.

Another aspect of the present invention provides a method for promoting aging of an animal (excluding humans), characterized by reducing the activity of Mre11 protein in a cell. More specifically, the method of inhibiting the activity of the Mre11 protein can deliver Mre11 protein inhibitor into the cell and / or inhibit the transcription of the Mre11 protein in the cell.

Another form of the present invention is to provide an aging agent characterized by including an intracellular Mre11 protein inhibitor.

Another form of the invention comprises the steps of treating any test substance with a test cell; Measuring the activity of Mre11 protein in the cells of the test cell; And it provides a aging agent screening method comprising the step of selecting a substance that reduces the activity of the intracellular Mre11 protein.

 Another aspect of the present invention is to provide a method for measuring the degree of aging, characterized by measuring the expression level of Mre11 protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to illustrate the present invention, but the content of the present invention is not limited by the Examples.

Example 1 Expression Decreased with Increased Age of Ku70 and Mre11 Proteins

Age-related decreases in DNA double-strand break (DSB) repair (Ref. 17) and age-dependent telomeres decrease have been known in human lymphocytes (Ref. 18). Expression of the DSB repair protein was confirmed to show age-related changes. To this end, blood was collected from 100 other volunteers, aged 20 to 80 years old in Seoul, Korea. All of these individuals were healthy and apparently without any signs of illness. Expression of the telomer binding proteins Ku70, Ku80, Sir2, TRF1, Mre11 was monitored by Western blot using a specific antibody that binds to telomeres.

human's CD4 Lymphocyte  detach

Human peripheral blood mononuclear cells (PBMCs) were obtained from 80 healthy volunteers from Jeju and 48 healthy volunteers between 20 and 80 years old in Seoul. All volunteers gave written informed consent for blood donations and fully understood the testing process. All test protocols were approved by the Institutional Review Board.

CD4-positive T cells were isolated from peripheral blood lymphocytes (PBL). Briefly, 50 ml of blood was collected from the tester and PBLs were separated by density gradient centrifugation using Ficoll-paque ^™ PLUS (Amersham Biosciences). CD4-positive T-cells were isolated using a negative depletion kit (Miltenyi Biotech). Purity of CD4 T cells was tested using a Fluorescence Activated Cell Sorter (FACS) with CellQuest Pro software (Becton Dickinson, NJ).

Western Blot  analysis

CD4-positive T cells were washed with phosphate-buffered saline (PBS) and, with a protein inhibitor cocktail (Roche), RIPA digestion buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 150 mM sodium chloride, 0.5% sodium deoxycholate and 0.1% SDS). Equal amounts (20 μg) of total protein were loaded on 10-12% SDS-PAGE. After electrophoresis, the protein was transferred to the nitrocellulose membrane. Proteins were determined by Western blotting luminescence by monoclonal rabbit anti-human Ku70 or anti-human Mre11 and horseradish peroxide-linked goat anti-rabbit Ig (Santa Cozu Biotechnology). Blots were stripped with stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol). Then again the reprobing blot was observed as monoclonal antibody anti-human β-actin. The density of the band was measured using a Huxix-bio-imaging analyzer BAS2500 (Fuji Photo Film).

Statistical analysis

Statistical characteristics for this study were expressed as n (%) or mean ± standard deviation, as appropriate. Student's tea test was used to compare the age distribution between control and longevity groups for the subjects in Jeju Island. The relationship between age for Ku70 or Mre11 for the study in Seoul was made using a simple first-order regression analysis. The effects of age and longevity groups on Ku70 were assessed using multiple regression analysis. Since there was no interaction effect between age and the group expressing Ku70, the main effect regression model was considered. Finally, the relationship between Ku70 and telomere length was examined by Pierce correlation analysis. All statistical analyzes were performed using SAS version 9.1 (Sars Institute Inc, NC, USA). The reported p-value is a two-side p-value, with p <0.05 considered to be statistically significant.

result

Of the proteins examined, the Ku70 and Mre11 proteins showed a marked decrease with age. As shown in FIG. 1, the level of Ku70 in people aged 60 and above was only 50% of people between 20 and 30 years old. The results of the first regression analysis of Ku70 (% of control) showed that the rate of Ku70 reduction was close to -1 (p = 0.0027; Table 2).

[Table 1] Characteristics of research subjects in Seoul

 Seoul area number  48 Age (years) *  51.5 ± 19.3

Mean ± Standard Deviation

[Table 2] Regression analysis of age for Mre11 and Ku70 in Seoul

Ku70 Mre11 variable* Coefficient Standard error p-value Coefficient Standard error P-value Intercept 108.50 13.48 <0.001 36.47 10.92 0.0017 age -0.78 0.25 0.0027 -0.47 0.20 0.3906

* R ² = 0.18 (for Ku70), 0.02 (for Mre11)

This shows that the expression of Ku70 is tightly regulated with age. Similarly, Mre11 expression decreased with age. However, expression of Mre11 was much later than the decrease of Ku70. All other telomere binding proteins Ku80, Sir2, TRF1 did not show changes in expression levels with age (data not shown). If the expression of Ku70 was related to the expression of Mre11.

As shown in Figure 2, the expression of Ku70 is directly related to that of Mre11. These data show that with age, CD4 lymphocytes gradually lose their ability to maintain high levels of Ku70 and Mre11, which will in part cause damage to DSB repair and telomere shortening.

The present invention can be used as a pharmaceutical composition or cosmetic composition for inhibiting aging by measuring the degree of aging of Mre11 protein, which is reduced as aging progresses, or supplemented with Mre11 protein into cells, and the amount of transcription of Mre11 in cells. It can increase the delay of the aging of the animal, it can also contribute to increase the productivity of the animal. In addition, it can promote aging by reducing the content of Mre11 protein, and can be used to induce aging by limiting the life of the animal when it is necessary to limit the life.

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Claims (11)

delete delete delete delete delete delete delete delete delete delete Aging degree measurement method characterized by measuring the expression level of Mre11 protein in animals except humans.

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